Abstract

Maintaining focus has been a critical but challenging issue in optical microscopy, particularly for microscopic imagingsystems currently used in biomedical research. During live cellimaging, environmental temperature fluctuations and other factors contribute to the unavoidable focus drift. For single molecular imaging and super resolution, focus drift can be significant even over short durations. The current commercial and experimental solutions are either optically complicated, expensive, or with limited axial resolution. Here, we present a simple autofocus solution based on low cost solid state laser and imaging sensor. By improving the optical train design and using real-time data analysis, improvement in axial resolution by approximately two orders of magnitudes over the focal depth of microscope objectives can be achieved. This solution has been tested for prolonged live cellimaging for fast ramping up in environmental chamber temperature and large daily swing in room temperature. In addition, this system can be used to spatial-temporally measure the surface for three-dimensional cell culture and tissue engineering, with flexibility that exceeds commercially available systems.

We thank Jianjiang Hu and Xiongfei Fu for assistance in experiments, Hongkai Wu and Yihua Zhao for providing the microlens array. This work is supported by a Research Grants Council of Hong Kong General Research Fund (No. 767711), a National Natural Science Foundation of China (NNSFC) Fund (No. 31270887) and University of Hong Kong seed funds for basic research (Nos. 104000978 and 104001876).